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Previously regarded as an intriguing photosynthetic curiosity, the occurrence of C4 and Crassulacean acid metabolism (CAM) photosynthesis within a single organism has recently emerged as a source of information for future biotechnological use. Among C4/CAM facultative species, Portulaca oleracea L. has been used as a model for biochemical and gene expression analysis of C4/CAM under field and laboratory conditions. In the present work, we focussed on developing molecular tools to facilitate functional genomics studies in this species, from the optimisation of RNA isolation protocols to a method for stable genetic transformation. Eleven variations of RNA extraction procedures were tested and compared for RNA quantity and quality. Also, 7 sample sets comprising total RNA from hormonal and abiotic stress treatments, distinct plant organs, leaf developmental stages, and subspecies were used to select, among 12 reference genes, the most stable reference genes for RT-qPCR analysis of each experimental condition. Furthermore, different explant sources, Agrobacterium tumefaciens strains, and regeneration and antibiotic selection media were tested in various combinations to optimise a protocol for stable genetic transformation of P. oleracea. Altogether, we provide essential tools for functional gene analysis in the context of C4/CAM photosynthesis, including an efficient RNA isolation method, preferred reference genes for RT-qPCR normalisation for a range of experimental conditions, and a protocol to produce P. oleracea stable transformants using A. tumefaciens.

Portulaca species can switch between C4 and crassulacean acid metabolism (CAM) depending on environmental conditions. However, the regulatory mechanisms behind this rare photosynthetic adaptation remain elusive. Using Portulaca oleracea as a model system, here we investigated the involvement of the circadian clock, plant hormones, and transcription factors in coordinating C4 and CAM gene expression. Free-running experiments in constant conditions suggested that C4 and CAM gene expression are intrinsically connected to the circadian clock. Detailed time-course, drought, and rewatering experiments revealed distinct time frames for CAM induction and reversion (days versus hours, respectively), which were accompanied by changes in abscisic acid (ABA) and cytokinin metabolism and signaling. Exogenous ABA and cytokinins were shown to promote and repress CAM expression in P. oleracea, respectively. Moreover, the drought-induced decline in C4 transcript levels was completely recovered upon cytokinin treatment. The ABA-regulated transcription factor genes HB7, NFYA7, NFYC9, TT8, and ARR12 were identified as likely candidate regulators of CAM induction following this approach, whereas NFYC4 and ARR9 were connected to C4 expression patterns. Therefore, we provide insights into the signaling events controlling C4–CAM transitions in response to water availability and over the day/night cycle, highlighting candidate genes for future functional studies in the context of facultative C4–CAM photosynthesis.

Portulaca oleracea is a C4 herb capable of performing CAM under drought stress. It is distributed worldwide and is either considered a polymorphic species or a complex of subspecies, due to its numerous morphological variations. We evaluated CAM plasticity within P. oleracea genotypes since the complexity surrounding this species may be reflected in intraspecific variations in photosynthetic behavior. Eleven subspecies of P. oleracea from distant geographical locations and one cultivar were morphologically and physiologically characterized. C4 and CAM photosynthesis were monitored in plants exposed to well-watered, droughted and rewatered treatments, and data obtained were compared among individual genotypes. All subspecies expressed CAM in a fully-reversible manner. Transcript abundance of C4–CAM signature genes was shown to be a useful indicator of the C4–CAM–C4 switches in all genotypes. C4-related genes were down-regulated and subsequently fully expressed upon drought and rewatering, respectively. CAM-marker genes followed the opposite pattern. A gradient of morphological traits and drought-induced nighttime malate accumulation was observed across genotypes. Therefore, different combinations of CAM expression levels, plant sizes and shapes are available within the P. oleracea complex, which can be a valuable tool in the context of C4/CAM photosynthesis research.

Previously regarded as an intriguing photosynthetic curiosity, the occurrence of C4 and Crassulacean acid metabolism (CAM) photosynthesis within a single organism has recently emerged as a source of information for future biotechnological use. Among C4/CAM facultative species, Portulaca oleracea L. has been used as a model for biochemical and gene expression analysis of C4/CAM under field and laboratory conditions. In the present work, we focussed on developing molecular tools to facilitate functional genomics studies in this species, from the optimisation of RNA isolation protocols to a method for stable genetic transformation. Eleven variations of RNA extraction procedures were tested and compared for RNA quantity and quality. Also, 7 sample sets comprising total RNA from hormonal and abiotic stress treatments, distinct plant organs, leaf developmental stages, and subspecies were used to select, among 12 reference genes, the most stable reference genes for RT-qPCR analysis of each experimental condition. Furthermore, different explant sources, Agrobacterium tumefaciens strains, and regeneration and antibiotic selection media were tested in various combinations to optimise a protocol for stable genetic transformation of P. oleracea. Altogether, we provide essential tools for functional gene analysis in the context of C4/CAM photosynthesis, including an efficient RNA isolation method, preferred reference genes for RT-qPCR normalisation for a range of experimental conditions, and a protocol to produce P. oleracea stable transformants using A. tumefaciens.

The SELF PRUNING (SP) gene is a key regulator of growth habit in tomato (Solanum lycopersicum). It is an ortholog of TERMINAL FLOWER1, a phosphatidylethanolamine-binding protein with antiflorigenic activity in Arabidopsis (Arabidopsis thaliana). A spontaneous loss-of-function mutation (sp) has been bred into several industrial tomato cultivars, as it produces a suite of pleiotropic effects that are favorable for mechanical harvesting, including determinate growth habit, short plant stature, and simultaneous fruit ripening. However, the physiological basis for these phenotypic differences has not been thoroughly explained. Here, we show that the sp mutation alters polar auxin transport as well as auxin responses, such as gravitropic curvature and elongation of excised hypocotyl segments. We also demonstrate that free auxin levels and auxin-regulated gene expression patterns are altered in sp mutants. Furthermore, diageotropica, a mutation in a gene encoding a cyclophilin A protein, appears to confer epistatic effects with sp. Our results indicate that SP affects the tomato growth habit at least in part by influencing auxin transport and responsiveness. These findings suggest potential novel targets that could be manipulated for controlling plant growth habit and improving productivity.

Though biochemically related, C4 and crassulacean acid metabolism (CAM) systems are expected to be incompatible. However, Portulaca species, including P. oleracea, operate C4 and CAM within a single leaf, and the mechanisms behind this unique photosynthetic arrangement remain largely unknown. Here, we employed RNA-seq to identify candidate genes involved exclusively or shared by C4 or CAM, and provided an in-depth characterization of their transcript abundance patterns during the drought-induced photosynthetic transitions in P. oleracea. Data revealed fewer candidate CAM-specific genes than those recruited to function in C4 . The putative CAM-specific genes were predominantly involved in nighttime primary carboxylation reactions and malate movement across the tonoplast. Analysis of gene transcript-level regulation and photosynthetic physiology indicated that C4 and CAM co-exist within a single P. oleracea leaf under mild drought conditions. Developmental and environmental cues were shown to regulate CAM expression in stems, whereas the shift from C4 to C4 -CAM hybrid photosynthesis in leaves was strictly under environmental control. Moreover, efficient starch turnover was identified as part of the metabolic adjustments required for CAM operation in both organs. These findings provide insights into C4 /CAM connectivity and compatibility, contributing to a deeper understanding of alternative ways to engineer CAM into C4 crop species.

Crassulacean acid metabolism (CAM) and the C4 pathway are photosynthetic adaptations that significantly improve plant water use efficiency. Both involve a prefixation of CO2 into acids prior to the carbon fixation via Rubisco and require refined regulatory mechanisms to synchronize reactions and minimize energy waste. C4 and CAM have been regarded as essentially incompatible, although the existence of C4–CAM facultative species challenges this assumption. Considering the potential emergence of drier areas in many parts of the globe due to climate change, understanding the compatibility between C4 and CAM holds enormous promise for future engineering of both photosynthetic adaptations in crop species. In this chapter, the major biochemical, anatomical, and molecular features of C4 and CAM are compared, and the stress signaling cascades controlling these syndromes are highlighted. The challenges and opportunities in combining the best of both photosynthetic adaptations into crops via genetic engineering approaches are also briefly discussed.

SummaryThe antiflorigenic signal SELF-PRUNING, which controls growth habit, exerts its effects through auxin transport, signaling and metabolism in tomato.AbstractTheSELF PRUNING(SP) gene is a key regulator of growth habit in tomato (Solanum lycopersicum). It is an ortholog ofTERMINAL FLOWER 1, a phosphatidyl-ethanolamine binding protein with anti-florigenic activity inArabidopsis thaliana. A spontaneous loss-of-functionspmutation has been bred into a large number of industrial tomato cultivars, as it produces a suite of pleiotropic effects that are favorable for mechanical harvesting, including determinate growth habit, short plant stature and simultaneous fruit ripening. However, the physiological basis for these phenotypic differences has not been thoroughly explained. Here, we show that thespmutation alters polar auxin transport as well as auxin responses such gravitropic curvature and elongation of excised hypocotyl segments. We further demonstrate that free auxin levels and auxin-regulated gene expression patterns are altered insp, with epistatic effects ofdiageotropica, a mutation in a cyclophilin A protein-encoding gene. Our results indicate that SP impacts growth habit in tomato, at least in part, via changes in auxin transport and responsiveness. These findings hint at novel targets that could be manipulated in the control of growth habit and productivity.

Made available in DSpace on 2018-12-11T16:44:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-02-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Floral anatomy and development of Saxofridericia aculeata Körn was studied in a comparative approach to contribute to the understanding of the family. Flowers at different developmental stages were analysed with light and scanning electron microscopy, and the nature of the exudate secreted by the floral trichomes was investigated by histochemical tests. The anatomical characteristics observed in S. aculeata flowers were compared with those from other Rapateaceae species by a cluster analysis (UPGMA). The dendrogram generated reflects the groupings that emerged in phylogenetic molecular analyses, highlighting the usefulness of floral anatomy for taxonomy and for the understanding of infrafamilial relationships. The exudate secreted by the trichomes has a polysaccharidic composition. Such trichomes (colleters) occur in the sepals, petals, filaments and around the gynoecium; they are initiated at mid-stage of floral development and are an apomorphy of the family. The flowers are pentacyclic, presenting three initially free sepals, petals, stamens and carpels that mature in a centripetal order. The connate portion of the corolla, which is also adnate to the stamens, has a late development by zonal growth. Gynoecium formation is a combination of postgenital and congenital fusion processes. Data on floral organogenesis of Rapateaceae are first reported here and support the early diverging position of the family in Poales, close to Bromeliaceae. Departamento de Botânica Instituto de Biociências Universidade Estadual Paulista (UNESP), C. Postal 199 Departamento de Botânica Instituto de Biociências Universidade Estadual Paulista (UNESP), C. Postal 199 CNPq: 168277/2014-0 FAPESP: 2014/22991-1